The present invention relates generally to viral vectors, and more specifically, to recombinant viral vectors which are capable of delivering vector constructs to susceptible target cells. These vector constructs are typically designed to deliver a gene product which is capable of activating a compound with little or no activity into an active product.
Although many bacterial diseases are, in general, easily treated with antibiotics, very few effective treatments exist for many viral, cancerous, and other diseases, including genetic diseases. For example, cancer now accounts for one-fifth of the total mortality in the United States, and is the second leading cause of death. Briefly, cancer is typically characterized by the uncontrolled division of a population of cells. This uncontrolled division typically leads to the formation of a tumor, which may subsequently metastasize to other sites.
Cancer, in general, represents a class of diseases which are very difficult to treat. For example, although primary solid tumors can generally be treated by surgical resection, a substantial number of patients that have solid tumors also possess micrometastases beyond the primary tumor site. If treated with surgery alone, many of these patients will experience recurrence of the cancer. Therefore, in addition to surgery many cancers are now also treated with cytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy. One difficulty with this approach however, is that radiotherapeutic and chemotherapeutic agents are toxic to normal tissues, and often create life-threatening side effects. In addition, these approaches often have extremely high failure/remission rates (up to 90% depending upon the type of cancer).
Various other therapies have thus been attempted, in an effort to bolster or augment an individual""s own immune system to eliminate cancer cells. Several such therapies have utilized bacterial or viral components as adjuvants, in order to stimulate the immune system to destroy the tumor cells. Examples of such components include BCG, endotoxin, mixed bacterial vaccines, interferons (xcex1, xcex2, and xcex3), interferon inducers (e.g., Brucella abortus, and various viruses), and thymic factors (e.g., thymosin fraction 5, and thymosin alpha-1) (see generally xe2x80x9cPrinciples of Cancer Biotherapy,xe2x80x9d Oldham (ed.), Raven Press, New York, 1987). Such agents have generally been useful as adjuvants and as nonspecific stimulants in animal tumor models, but have not yet proved to be generally effective in humans.
Lymphokines have also been utilized in the treatment of cancer. Briefly, lymphokines are secreted by a variety of cells, and generally have an effect on specific cells in the generation of an immune response. Examples of lymphokines include Interleukins (IL)-1, -2, -3, and -4, as well as colony stimulating factors such as G-CSF, GM-CSF, and M-CSF. Recently, one group has utilized IL-2 to stimulate peripheral blood cells in order to expand and produce large quantities of cells which are cytotoxic to tumor cells (Rosenberg et al., N. Engl. J. Med. 313:1485-1492, 1985).
Others have suggested the use of antibody-mediated anti-cancer therapies. Briefly, antibodies may be developed which recognize certain cell surface antigens that are either unique, or more prevalent on cancer cells compared to normal cells. These antibodies, or xe2x80x9cmagic bullets,xe2x80x9d may be utilized either alone or conjugated with a toxin in order to specifically target and kill tumor cells (Dillman, xe2x80x9cAntibody Therapy,xe2x80x9d Principles of Cancer Biotherapy, Oldham (ed.), Raven Press, Ltd., New York, 1987). For example, Ball et al. (Blood 62:1203-1210, 1983) treated several patients with acute myelogenous leukemia with one or more of several monoclonal antibodies specific for the leukemia, resulting in a marked decrease in circulating leukemia cells during treatment. Similarly, others have utilized toxin-conjugated antibodies therapeutically to treat a variety of tumors, including, for example, melanomas, colorectal carcinomas, prostate carcinomas, breast carcinomas, and lung carcinomas (see Dillman, supra). One difficulty however, is that most monoclonal antibodies are of murine origin, and thus hypersensitivity against the murine antibody may limit its efficacy, particularly after repeated therapies. Common side effects include fever, sweats and chills, skin rashes, arthritis, and nerve palsies.
Therefore cancer has, as a general rule, been very difficult to treat utilizing either conventional or experimental pharmaceutical compositions.
Likewise, viral diseases have been very difficult to treat with conventional pharmaceutical compositions. In general, such pharmaceuticals have lacked specificity, exhibit a high overall toxicity, and have generally been found to be therapeutically ineffective.
Other techniques which have been developed for treating viral diseases involve the elicitation of an immune response to a pathogenic agent (i.e., the virus) through the administration of a noninfectious form of the virus (such as a killed virus), thereby providing antigens which act as an immunostimulant. Such an approach has proved useful for certain viruses (e.g., polio) but not for other viruses (e.g., HIV).
A more recent approach for treating viral diseases, such as acquired immunodeficiency syndrome (AIDS) and related disorders, involves blocking receptors on cells susceptible to infection by HIV from receiving or forming a complex with viral envelope proteins. For example, Lifson et al. (Science 232:1123-1127, 1986) demonstrated that antibodies to CD4 (T4) receptors inhibited cell fusion (syncytia) between infected and noninfected CD4 presenting cells invitro. A similar CD4 blocking effect using monoclonal antibodies has been suggested by McDougal et al. (Science 231:382-385, 1986). Alternatively, Pert et al. (Proc. Natl. Acad. Sci. USA 83:9254-9258, 1986) reported the use of synthetic peptides to bind T4 receptors and block HIV infection of human T-cells, and Lifson et al. (J. Exp. Med. 164:2101, 1986) reported blocking both syncytia and virus/T4 cell fusion by using a lectin which interacts with a viral envelope glycoprotein, thereby blocking it from being received by CD4 receptors.
An alternative technique for inhibiting a pathogenic agent, such as a virus (which transcribes RNA), is to provide antisense RNA which complements at least a portion of the transcribed RNA, thereby inhibiting translation (To et al., Mol. Cell. Biol. 6:758, 1986).
A major shortcoming, however, of the techniques described above is that they do not readily lend themselves to control the time, location or extent to which a drug, antigen, blocking agent or antisense RNA is utilized. In particular, since the above techniques require exogenous application of the treatment agent (i.e., exogenous to the sample in an in vitro situation), they are not directly responsive to the presence of the pathogenic agent. For example, it may be desirable to have an immunostimulant expressed in increased amounts immediately following infection by the pathogenic agent. In addition, in the case of antisense RNA, large amounts would be required for useful therapy in an animal, which under current techniques would be administered without regard to the location at which it is actually needed, that is, in cells infected with the pathogenic agent.
As an alternative to exogenous application, techniques have been suggested for producing treatment agents endogenously. More specifically, proteins expressed from viral vectors based on DNA viruses, such as adenovirus, simian virus 40, bovine papilloma, and vaccinia viruses, have been investigated. By way of example, Panicali et al. (Proc. Natl. Acad. Sci. USA 80:5364, 1983) introduced influenza virus hemagglutinin and hepatitis B surface antigens into the vaccinia genome and infected animals with the virus particles produced from such recombinant genes. Following infection, the animals acquired immunity to both the vaccinia virus and the hepatitis B antigen.
A number of difficulties however have been experienced to date with viral vectors based upon DNA viruses. These difficulties include: (a) the production of other viral proteins which may lead to pathogenesis or the suppression of the desired protein; (b) the capacity of the vector to uncontrollably replicate in the host, and the pathogenic effect of such uncontrolled replication; (c) the presence of wild-type virus which may lead to viremia; and (d) the transitory nature of expression in these systems. These difficulties have virtually precluded the use of viral vectors based on DNA viruses in the treatment of viral, cancerous, and other nonbacterial diseases, including genetic diseases.
Due to the nontransitory nature of their expression in infected target cells, retroviruses have been suggested as a useful vehicle for the treatment of genetic diseases (for example, see F. Ledley, The Journal of Pediatrics 110:1, 1987). However, in view of a number of problems, the use of retroviruses in the treatment of genetic diseases has not yet been widely accepted. Such problems relate to: (a) the apparent need to infect a large number of cells in inaccessible tissues (e.g. brain); (b) the need to cause these vectors to express in a very controlled and permanent fashion; (c) the lack of cloned genes; (d) the irreversible damage to tissue and organs due to metabolic abnormalities; and (e) the availability of other partially effective therapies in certain instances.
The present invention provides novel compositions and methods for treating a variety of diseases (e.g., viral diseases, cancer, genetic diseases and others), and further provides other, related advantages.
Briefly stated, the present invention provides recombinant viral vectors and methods of using such vectors for the treatment of a wide variety of pathogenic agents. Within one aspect of the invention, recombinant viral vectors are provided carrying a vector construct which directs the expression of a gene product capable of activating an otherwise inactive precursor into an active inhibitor of a pathogenic agent. Within one embodiment of the invention, the pathogenic agent is a virus-infected cell. Within another embodiment, the gene product is Herpes Simplex Virus Thymidine Kinase or Varicella Zoster Virus Thymidine Kinase. Within yet another embodiment, the inactive precursor is AZT.
Within another aspect of the present invention, recombinant viral vectors are provided carrying a vector construct which directs the expression of a gene product that activates a compound with little or no cytotoxicity into a toxic product. Within other aspects, recombinant viral vectors are provided carrying a vector construct which directs the expression of a gene product that activates a compound with little or no cytotoxicity into a toxic product in the presence of a pathogenic agent, thereby affecting localized therapy to the pathogenic agent. Within one embodiment, the gene product is Herpes Simplex Virus Thymidine Kinase, or Varicella Zoster Virus Thymidine Kinase. Within other embodiments, the gene product is selected from the group consisting of E. coli guanine phosphoribosyl transferase, alkaline phosphatase, fungal cytosine deaminase, carboxypeptidease G2, and Penicillin-V amidase. Within yet other embodiments, the pathogenic agent is a virus-infected cell, a cell infected with bacteria, a tumor cell, or a cell infected with a parasite.
Within other aspects of the invention, recombinant viral vectors are provided which direct the expression of a protein that is toxic upon processing or modification by a protein derived from a pathogenic agent. Within one embodiment, the protein which is toxic upon processing or modification is proricin.
Within yet another aspect of the invention, recombinant viral vectors are provided carrying a vector construct comprising a cytotoxic gene under the transcriptional control of an event-specific promoter, such that upon activation of the event-specific promoter the cytotoxic gene is expressed. Within various embodiments, the event-specific promoter is a cellular thymidine kinase promoter, or a thymidylate synthase promoter. Within another embodiment, the event-specific promoter is activated by a hormone. Within yet other embodiments, the cytotoxic gene is selected from the group consisting of ricin, abrin, diptheria toxin, cholera toxin, gelonin, pokeweed, antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A.
Within another aspect of the present invention, recombinant viral vectors are provided comprising a cytotoxic gene under the transcriptional control of a tissue-specific promoter, such that upon activation of the tissue-specific promoter the cytotoxic gene is expressed. Within various embodiments, the tissue-specific promoter is the phosphoenopyruvate carboxykinase (PEPCH) promoter, HER2/neu promoter; casein promoter, IgG promoter, Chorionic Embryonic Antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, tyrosine hydroxylase promoter, albumin promoter, alphafetoprotein promoter, acetyl-choline receptor promoter, alcohol dehydrogenase promoter, xcex1 or xcex2 globin promoter, T-cell receptor promoter, the osteocalcin promoter the IL-2 promoter, IL-2 receptor promoter, whey (wap) promoter, and the MHC Class II promoter.
Within yet another aspect of the present invention, viral vectors are provided carrying a vector construct comprising a cytotoxic gene under the transcriptional control of both an event-specific promoter and a tissue-specific promoter, such that the cytotoxic gene is maximally expressed only upon activation of both the event-specific promoter and the tissue-specific promoter. Representative event-specific and tissue-specific promoters have been discussed above. Within one preferred embodiment, the event-specific promoter is thymidine kinase, and the tissue-specific promoter is selected from the group consisting of the casein promoter and the HER2/neu promoter.
Within other aspects of the invention, viral vectors similar to those described above are provided, except that, in place of (or in addition to) the vector construct which directs the expression of acytotoxic gene, the viral vector carries a vector construct which directs the expression of a gene product that activates a compound with little or no cytotoxicity into a toxic product.
Within other aspects of the present invention, the vector contructs described above may also direct the expression of additional non-vector derived genes. Within one embodiment the non-vector derived gene encodes a protein, such as an immune accessory molecule. Representative examples of immune accessory molecules include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, B7, B7-2, GM-CSF, CD3, ICAM-1, xcex2-microglobulin, LFA-3, HLA Class I, and HLA Class II molecules. Within one preferred embodiment, the protein is gamma-interferon.
Within other aspects of the present invention, methods are provided for inhibiting or destroying pathogenic agents in a warm-blooded animal, comprising administering to a warm-blooded animal a recombinant viral vector as described above, such that the pathogenic agent is inhibited or destroyed. As utilized herein, it should be understood that the term xe2x80x9cdestroyedxe2x80x9d refers to the destruction of cells which are responsible for a disease state, which destruction may result in only partial destruction of the disease (e.g., tumors may be only partially destroyed). Within various embodiments, the recombinant viral vector is administered in vivo, or alternatively, ex vivo. Within yet other embodiments, the pathogenic agent is a virus-infected cell, a cell infected with bacteria, or a tumor cell.
Within other aspects of the present invention, producer cells are provided which generate a recombinant viral vector as described above. Within another aspect, methods are provided for destroying pathogenic agents in a warm blooded animal, comprising administering to the animal such producer cells, in order to destroy the pathogenic agent.
Within another aspect of the present invention, methods are provided for destroying a pathogenic agent in a warm blooded animal, comprising the step of administering to the warm blooded animal nucleic acids which encode a gene product that activates a compound with little or no cytotoxicity into a toxic product such that the pathogenic agent is destroyed.
Within yet another aspect of the invention, pharmaceutical compositions are provided, comprising a recombinant viral vector as described above, in combination with a pharmaceutically acceptable carrier or diluent.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, a number of patents, patent applications, and other publications are disclosed below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.